This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Brassinosteriods (BRs) are steroid hormones with important roles in plants. BR signal is perceived by the cell-surface receptor kinase BRI1, which initiates a cascade of protein phosphorylation leading to nuclear gene expression and cellular responses. Downstream BR signaling involves the cytoplasmic GSK3/SHAGGY-like kinase BIN2. BIN2 phosphorylates the transcription factors BZR1 and BZR2/BES1, and this phosphorylation inhibits their DNA binding and nuclear localization and promotes their degradation by the proteasome. The goal of this study is to understand BR signal transduction at the biochemical and proteomic levels. First, we will identify the BIN2-phosphorylation sites of BZR1 using mass spectrometry (MS). The importance of some of the phosphorylation sites will be further tested by site-directed-mutagenesis and transgenic experiments. Second, we will identify the BZR1-interacting proteins using tandem affinity purification and mass spectrometry analysis. Third, in order to understand how BR signaling regulates BIN2 activity, we will purify BIN2-interacting proteins and identify them using MS analysis. The functions of the identified proteins will be studied using genetic and transgenic experiments. This research project will advance our understanding of the molecular mechanism for steroid responses in plants, which will have broad implications in our understanding of steroid actions in general.